延缓衰老基因PPF1转化水稻的初步研究

利用农杆菌介导法,把短日豌豆花后特异表达、具有延缓衰老作用的基因PPF1（post-floral-specific gene expressed in short-day-grown G2 pea）转入水稻,以期获得延缓衰老的转基因植株。约970块未成熟胚性愈伤组织与农杆菌共培养后,得到了约280块抗性愈伤组织,从中分化出抗性绿苗54株。PCR和Southern blotting检测结果表     

Development of elite rice restorer lines in the genetic background of R022 possessing tolerance to brown planthopper, stem borer, leaf folder and herbicide through marker-assisted breeding

Rice leaf folder, stem borer and brown planthopper (BPH) are the most devastating rice insect pests. Developing and planting insect-resistant rice varieties is the most economical and effective measure for controlling these pests. BPH can be controlled with native BPH-resistance genes in rice, while at present rice leaf folder and stem borer can only be controlled through planting transgenic Bt rice. In this study, the breeding of a new restorer line KR022 possessing stacked BPH-resistance genes Bph14 and Bph15, Bt gene cry1C and glufosinate-resistance gene bar, is reported for the first time. A rice restorer line R022 with BPH-resistance genes Bph14 and Bph15 was used as a recurrent parent to cross with the transgenic rice T1C-19 of cry1C and bar genes during the breeding process. The restorer line KR022 was developed from the backcross populations of R022 and T1C-19 through molecular marker-assisted selection and glufosinate-resistance selection. The cry1C and bar genes were found to integrate on chromosome 11 of KR022, and the genome recovery of KR022 was up to 95.8 % of the R022 genome. The quantification of Cry1C protein expression showed that it was expressed at different levels in the leaf, stem, panicle, endosperm, and root of KR022 and its hybrid rice. The insect-resistance evaluation indicated that KR022 and its hybrid rice had good resistance to rice leaf folder and stem borer, both in laboratory settings and in the field. Furthermore, they exhibited increased resistance to BPH at both the seedling and mature stage. The field trial showed there was no significant difference in key agronomic traits between KR022 and its recurrent parent R022, and four hybrids from KR022 yield much higher than the control II-You 838. Moreover, KR022 and its hybrid rice were found to have resistance to the herbicide glufosinate. These results demonstrate that KR022 is effective as a rice restorer line for the breeding of “green super rice”, possessing multiple tolerances to rice BPH, stem borer, leaf folder and glufosinate.     

The transformation of the photo-thermo sensitive genic male-sterile line 261S of rice via an expression vector containing the anti-Waxy gene

Transgenic photo-thermo sensitive genic male sterility Oryza sativa L. cv. “261S” plants with the anti-Waxy gene were successfully obtained using an Agrobacterium tumefaciens-mediated co-transformation method. Marker-free homozygous transgenic lines with the anti-Waxy gene were obtained. The setting seed rates of the transgenic plants via self-pollination or via crossing with the restorer line WX99075 rice and the 1000-grain weight of the transgenic plants and the F₂ hybrid seeds obtained by crossing the transgenic or non-transgenic plants with the restorer line WX99075 rice, and the number of panicles of the transgenic plants and yields of the F₂ hybrid rice, were analysed. Quality indexes of the transgenic plants and of the F₂ hybrid seeds were analysed. Our researches results indicate that hybrid female and hybrid descendant edibility could be improved via the introduction of the anti-Waxy gene, but the grain yields of the reserve seeds via self-pollination of the transgenic photo-thermo sensitive genic sterile lines and of the hybrid rice were not affected.     

Breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer and herbicide

In this paper, we described the breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer (SSB) and herbicide by conventional crossing of two transgenic parental lines transformed independently with different genes. Two stable transgenic rice lines used as donor parents were developed, one was Zhongguo91 which contained cry1Ab gene (for insect resistance) and bar gene (for tolerance of herbicide), and the other was Yujing6 which contained Xa21 gene (resistance to bacterial blight). The elite restorer line Hui773 was used as recipient and crossed with the two stable transgenic rice lines. Then five successive backcrosses were made using Hui773 as recurrent parent. Two rice elite restorers, T773-1 expressing cry1Ab and bar genes and T773-2 expressing Xa21 gene, were obtained, which were confirmed by PCR analysis and testing selectable marker genes in the hybrid progenies. The cross was made between T773-1 and T773-2 to select stable restorer line carrying Xa21, cry1Ab and bar genes. Finally, we obtained transgenic restorer line T773 with good agronomic traits and obvious multiple resistance to Xanthomonas oryzae pv. oryzae, striped stem borer (Chilo suppressalis) and herbicide. The hybrid F₁ generation produced from the cross between transgenic restorer line T773 and a corresponding male sterile line Zaohua2A maintained obvious resistance to rice bacterial blight, rice leaffolder and striped stem borer, and showed significant heterosis. Our results indicate that it is feasible to develop transgenic hybrid rice cultivar through breeding transgenic restorer lines.     

Resistance to the cabbage root fly, Delia radicum, within Brassica fruticulosa

Two transgenic Bt rice lines, KMD1 and KMD2, both containing a synthetic cry1Ab gene from Bt, were crossed with conventional rice varieties. The inheritance of resistance to SSB of KMD1 and KMD2was investigated through LSB and field examination of their progenies, e.g. F₁, BC₁ and F₂ populations. In LSBs, 100.0% of newly hatched SSB larvae died on the second day after feeding on leaf tissues of F₁ and GUS positive BC₁ plants, of which the area of leaf tissues consumed by SSB is also similar to that of transgenic parents. These results imply that the resistance of Bt rice to SSB is dominantly controlled and could be easily exploited in hybrid rice production. Field evaluation showed that segregation ratios for SSB resistance to susceptibility in BC₁ populations fit the ratio of 1:1, which was also confirmed by LSBs. However, in F₂ populations, the ratio was significantly smaller than 3:1 for resistant to susceptible plants in all 6 indica × japonica (KMD1 and KMD2) crosses, though it fitted 3:1 in all 4 japonica × japonica crosses. The results implied that the resistance of Bt rice to SSB was controlled by a dominant gene which was present in a homozygous condition in both KMD1 and KMD2, but the inheritance could be affected by other factors. Assays for Cry1Ab protein showed that, in most crosses, the content of Cry1Ab is significantly higher in leaves of GUS positive F₁, BC₁ and F₂ plants than that in transgenic Bt parent plants, which accounts for the high resistance observed in these plants to SSB. This revised version was published online in July 2006 with corrections to the Cover Date.     

转Xa23基因水稻的白叶枯病抗性及其遗传分析

在分离克隆抗白叶枯病基因Xa23研究中获得大量转基因水稻材料。为了系统研究转Xa23基因水稻的抗病稳定性和遗传模式, 本文通过逐株进行抗白叶枯病接种鉴定、PCR和Southern blot分子检测, 对一批转Xa23基因水稻植株进行了T₀代到T₂代的跟踪分析。结果表明, Xa23基因的整合和表达, 使感病受体品种牡丹江8号获得抗病性。由于Xa23基因插入受体基因组的位点不同, 同是单拷贝插入的转基因T₀代抗病植株, 其抗病程度有明显差异。T₀代植株的抗病程度, 可以准确、稳定地遗传到T₁代和T₂代。单拷贝转基因植株分离群体的抗感植株分离比接近3∶1, 表明转Xa23基因遵循孟德尔单基因遗传模式。已获得2个纯合的单拷贝转基因抗病株系, 它们的抗病程度稍有差别, 将用于外源基因插入位置效应分析和杂交稻抗病育种。     

Senescence of Flag Leaf and Glume in Rice: Role of Grains during Source-Sink Modification by Physical and Chemical Means

Senescence of flag leaf and glume of rice ( Oryza sativa L. cv. Ratna ) at different source sink ratios, through manipulating the plants by physical and chemical means, were analyzed. As judged from the chlorophyll retention capacity, induction of sterility deferred senescence of flag leaf and glume at both grain filling and post grain maturation period. Senescence of the glumes was promoted in the plants whose panicles were darkened. The time needed for achieving maximum 1000 grain dry weight was quickened, the harvest index (HI) was lowered and the flag leaf senescence was delayed by such manipulation. Spraying of urea deferred senescence of the flag leaf as well as increased 1000 grain dry weight and HI. The time taken for achieving maximum 1000 grain dry weight was, however, delayed. Senescence of glumes was promoted by α-HPMS spray, while the same was delayed by PMA spray on the whole plant. Both the chemicals deferred flag leaf senescence and decreased 1000 grain dry weight and HI. Senescence of the flag leaf at post grain filling period was also delayed by the above physical and chemical manipulations compared to that of control, the highest delaying effect was recorded in the flag leaf of sterile plant.
It is concluded that nutrient drainage is the primary cause of nonsequential senescence of the flag leaf of rice (cv. Ratna ). The involvement of senescence signal if any, is perhaps of secondary importance.     

利用花粉管通道法将叶片衰老抑制基因PSAG12-IPT导入普通小麦的研究

利用花粉管通道法将叶片衰老抑制基因PSAG12-IPT导入普通小麦品种西农1376,经PCR﹑GUS组织化学染色﹑点杂交和Southern杂交分析,证明带有特异启动子的目的基因已整合到5个植株的基因组中,且在大部分转基因植株中能够稳定遗传。通过叶片细胞分裂素含量﹑叶绿素含量﹑叶片衰老进程及农艺性状分析,初步证明PSAG12-IPT基因在     

Expression of a lipoxygenase encoding gene (BoLOX1) during postharvest senescence of broccoli

Lipoxygenases (LOX) belong to a large family of plant enzymes that catalyze the hydroperoxidation of polyunsaturated fatty acids. Most of them are expressed during senescence and contribute to membrane deterioration and biosynthesis of jasmonic acid, a known senescence enhancer. In this work, we cloned a fragment of a gene encoding a LOX from broccoli (BoLOX1). The analysis of the sequence revealed that BoLOX1 is closely related to other LOX from higher plants. Furthermore, we analyzed the expression of BoLOX1 and detected a larger increase during postharvest senescence. A slight increase of total lipoxygenase activity was also found during senescence. In other sets of experiments, broccoli heads were treated with plant hormones, such as cytokinin and ethylene, as a way to assess the effect of such compounds on the expression of BoLOX1. Cytokinin treatment delayed the increase of BoLOX1 expression and lipoxygenase activity whereas ethylene accelerated both processes. Also, several postharvest treatments were applied in order to delay senescence in broccoli florets and to evaluate their effects on BoLOX1 expression. Samples treated with modified atmosphere, hot air, UV-C or white light showed a delay in chlorophyll degradation and degreening. In most cases, the treatments also delayed the increase of BoLOX1 expression, reaffirming the relationship between the expression of this gene and senescence. However, treatments like modified atmospheres and visible light markedly increased lipoxygenase activity, which suggests a lack of correlation between BoLOX1 expression and lipoxygenase activity.     

Close linkage of a blast resistance gene,Pias(t), with a bacterial leaf blight resistance gene,Xa1-as(t), in a rice cultivar ‘Asominori’

It has long been known that a bacterial leaf blight-resistant line in rice obtained from a crossing using ‘Asominori’ as a resistant parent also has resistance to blast, but a blast resistance gene in ‘Asominori’ has not been investigated in detail. In the present study, a blast resistance gene in ‘Asominori’, tentatively named Pias(t), was revealed to be located within 162-kb region between DNA markers YX4-3 and NX4-1 on chromosome 4 and to be linked with an ‘Asominori’ allele of the bacterial leaf blight resistance gene Xa1, tentatively named Xa1-as(t). An ‘Asominori’ allele of Pias(t) was found to be dominant and difference of disease severity between lines having the ‘Asominori’ allele of Pias(t) and those without it was 1.2 in disease index from 0 to 10. Pias(t) was also closely linked with the Ph gene controlling phenol reaction, suggesting the possibility of successful selection of blast resistance using the phenol reaction. Since blast-resistant commercial cultivars have been developed using ‘Asominori’ as a parent, Pias(t) is considered to be a useful gene in rice breeding for blast resistance.     

A treatment to improve the vase life of cut tulips: Effects on tepal senescence, tepal abscission, leaf yellowing and stem elongation

The vase life of cut tulips (Tulipa spp.) is limited by a combination of leaf yellowing, tepal senescence, and tepal abscission. In many cultivars, moreover, high rates of stem elongation result in stem bending during vase life. In tests with the cvs. Apeldoorn and Frappant, stem bending could be prevented by treatment with ethylene or ethephon. However, these treatments resulted in poor flower opening. The ethephon treatment also resulted in precocious tepal abscission. The negative effect of ethephon on flower opening was overcome by a treatment with gibberellic acid (GA₃). GA₃ also somewhat delayed early leaf yellowing, but did not prevent the early tepal abscission induced by ethephon. The latter problem was overcome by a treatment with benzyladenine (BA). In addition, BA effectively delayed leaf yellowing and also delayed tepal senescence. However, BA produced browning of the lower stem end. This was prevented by the inclusion of calcium ions in the solution. The combination of chemicals (ethephon, GA₃, BA, and calcium ions) was similarly successful in a large number of other tulip cultivars tested. After adaptation of the concentrations the four chemicals were also effective if given as a pulse treatment shortly after harvest.     

Development of cleaved amplified polymorphic sequence (CAPS) markers linked to a green rice leafhopper resistance gene, Grh3(t)

The chromosomal location of the resistance gene for green rice leafhopper (GRLH), an injurious insect for rice, has been determined and RFLP markers closely linked to this gene have been identified. The susceptible japonica rice variety ‘Nipponbare’ was crossed with a resistant japonica rice line ‘Aichi42’, in which green rice leaf hopper resistance had been introduced from an indica variety ‘Rantaj‐emas2’, and the 100 F₂ plants obtained were used for linkage analysis. The green rice leafhopper resistance gene, Grh3(t), was mapped between RFLP markers C288B and C133A on chromosome 6 and co‐segregated with C81. Of the RFLP markers tightly linked to Grh3(t), C81 was converted to a SCAR marker and C133A to a cleaved amplified polymorphic sequence marker that could distinguish the heterozygous genotype to establish an effective marker‐aided selection system for the GRLH resistance gene.     

水稻叶片淀粉积累及早衰突变体esl9的鉴定与基因定位

利用EMS诱变籼型三系保持系西农1B,获得了一个新的水稻叶片淀粉过度积累而导致的早衰突变体esl9(early senescence leaf 9)。该突变体苗期叶片呈淡绿色,分蘖期开始除心叶外的叶片从叶尖开始黄化衰老,逐渐延伸至叶中上部,基部保持绿色,该性状一直持续到成熟。与野生型相比,esl9叶片光合色素含量下降,O_2~-、·OH和H_2O_2等活性氧含量上升,保护酶系统中SOD和CAT活性降低。组织化学分析表明,esl9叶片中积累了大量的淀粉颗粒,淀粉含量显著上升;qRT-PCR结果显示,淀粉合成途径相关基因上调,磷酸丙糖(TP)分配途径基因下调,推测基因突变可能改变了TP的分配途径,导致叶片过度积累淀粉,破坏叶绿体结构,光合系统受阻,活性氧增多,引起叶片黄化衰老。遗传分析表明该突变体受一对显性核基因调控,ESL9位于第11染色体标记S11-110与S11-87之间,物理距离为304.9 kb,这为进一步基因克隆和功能研究奠定了基础。     

水稻早衰突变体esl5的鉴定及其基因精细定位

叶片早衰直接影响作物产量和品质, 鉴定早衰突变体、图位克隆调控基因对于研究植物衰老机理具有重要的意义。以甲基磺酸乙酯诱变水稻籼型恢复系缙恢10号, 获得一个早衰突变体esl5 (early senescent leaf mutant 5), 本文对其进行了形态鉴定、细胞学观察、理化分析和基因定位等研究。结果表明, 与野生型相比, esl5的苗期叶片正常, 分蘖期呈黄绿色, 孕穗期开始叶片中上部逐渐黄化衰老; 衰老部位的细胞结构异常, 细胞膜降解, 叶绿体基质片层疏松、排列不规则, 光合色素含量和光合速率极显著下降。此外, esl5的·OH和H₂O₂含量极显著升高, SOD和CAT的活性则极显著降低。与野生型相比, esl5的生育期延长了20 d左右, 千粒重显著增加, 穗粒数、实粒数和结实率则显著降低。esl5受1对隐性核基因调控, 精细定位在第3染色体Indel标记Indel03-1和Indel03-2之间83.4 kb的物理范围内, 包含11个注释基因, 这为ESL5的克隆和功能研究奠定了基础, 也有利于水稻品种的遗传改良。     

QTL analysis on rice grain appearance quality,as exemplifying the typical events of transgenic or backcrossing breeding

Rice grain shape and yield are usually controlled by multiple quantitative trait loci (QTL). This study used a set of F_9–10 recombinant inbred lines (RILs) derived from a cross of Huahui 3 (Bt/Xa21) and Zhongguoxiangdao, and detected 27 QTLs on ten rice chromosomes. Among them, twelve QTLs responsive for grain shape/ or yield were mostly reproducibly detected and had not yet been reported before. Interestingly, the two known genes involved in the materials, with one insect-resistant Bt gene, and the other disease-resistant Xa21 gene, were found to closely link the QTLs responsive for grain shape and weight. The Bt fragment insertion was firstly mapped on the chromosome 10 in Huahui 3 and may disrupt grain-related QTLs resulting in weaker yield performance in transgenic plants. The introgression of Xa21 gene by backcrossing from donor material into receptor Minghui 63 may also contain a donor linkage drag which included minor-effect QTL alleles positively affecting grain shape and yield. The QTL analysis on rice grain appearance quality exemplified the typical events of transgenic or backcrossing breeding. The QTL findings in this study will in the future facilitate the gene isolation and breeding application for improvement of rice grain shape and yield.     

Two novel QTLs regulate internode elongation in deepwater rice during the early vegetative stage

Deepwater rice possesses internode elongation ability to avoid drowning under deepwater conditions. Previous studies identified three QTLs regulating internode elongation ability on chromosomes 1, 3 and 12 using different populations. However, these QTLs only induce internode elongation in response to deepwater conditions from the 7-leaf stage and not during the early leaf stage. In this study, we detected two novel QTLs, qTIL2 and qTIL4 regulating deepwater response at the early leaf stage using an F₂ population derived from the cross between NIL1-3-12 carrying the three QTLs regulating deepwater response in T65 (O. sativa ssp. japonica) genetic background and C9285 (O. sativa ssp. indica, deepwater rice). Plants of the BC₂F₂ population derived from NIL1-3-12/C9285 and the RILs of T65/Bhadua (O. sativa ssp. indica, deepwater rice) possessing these QTLs as well as the three QTLs previously identified also showed internode elongation during the early leaf stage. These results indicate that qTIL2 and qTIL4 regulate early internode elongation and function in coordination with the three major QTLs under deepwater conditions. The results presented here would not only help define the mechanism of deepwater response in rice but also contribute in the breeding of deepwater tolerant rice that is adapted to various water depths.     

Examination and genetic analysis of a yellow-green leaf mutant a269 of sweetpotato

The genetic behaviour of a yellow-green leaf sweetpotato mutant, a269, was analysed to prepare for genetic mapping and cloning of this mutant and to elucidate the regulation of its molecular mechanisms. The phenotypic stability of the mutant was observed under natural conditions, and photosynthetic pigments in mature leaves were measured. GN1323 and a269 were crossed and backcrossed. Genetic behaviour was assessed using phenotypic expression and segregation in F₁, BC₁ and BC₂. The a269 leaves were yellow-green at all stages after germination. The thylakoid lamellae of a269 were disorganized, had incomplete grana and were fewer compared with GN1323. The direct and reciprocal F₁ populations had normal leaf colour, whereas the BC₂ populations had yellow-green leaves. Segregation of plants with normal- and yellow-green leaf colour in the four BC₁ populations satisfied the backcross segregation ratio of 11 : 1 for auto-allohexaploids. The a269 mutant is stable, and its trait is controlled by one recessive nuclear locus.     

Comparative Study of CaMsrB2 Gene Containing Drought‐Tolerant Transgenic Rice (Oryza sativa L.) and Non‐Transgenic Counterpart

Concerns regarding the safety of transgenic foods have been raised because of possibility of undesirable effects development during genetic engineering. Analysis of phenotypic traits can increase the likelihoods of identifying those unintended effects in dietary composition of the GM crops. Objective of this study was to compare the transgenic lines with their non‐transgenic counterpart. Different vegetative and reproductive traits as well as antioxidant properties were considered to evaluate the transgenic (HV8 and HV23) lines containing CaMsrB2 gene and their non‐transgenic (Ilmi) parent line. Grain size and weight, seed germination, root length, root and shoot dry weight, length and width of flag leaf, plant height, and ligule, stamen and carpel length were not significantly different. Onset and completion of heading in each line occurred almost during the same period. The antioxidant properties in terms of DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging activity and polyphenol content were not statistically different under same treatment condition. The results suggested that the transgenic rice lines containing CaMsrB2 gene were equivalent to their non‐transgenic counterpart without any visible unintended effects.     

水稻蛋白二硫键异构酶基因沉默载体构建及其转基因后代的高温结实特性分析

采用RT-PCR法亚克隆了PDI基因保守区内450 bp的靶标序列作为干扰区段, 构建了含有内含子hpRNA (ihpRNA)的双元表达载体pTCK303-RiOsPDI, 经农杆菌介导转化日本晴, 获得转基因植株; 通过在T₀代对其潮霉素(Hyg)抗性基因的PCR鉴定, 确定携带有干扰片段的T-DNA区已整合到水稻基因组中, 且在转基因T₁代符合3∶1的分离模式。半定量PCR和荧光定量PCR的检测结果表明, PDI基因沉默转基因阳性植株不同器官中的PDI表达量均显著降低, 尤其是其籽粒中表达量较微, 几乎能引起靶基因80%左右沉默。对转基因T₂代植株的高温结实特性和籽粒理化品质的检测结果, PDI基因沉默会引起高温胁迫处理下结实率的大幅度降低, 耐热性显著下降, 但其在常温处理下的结实率与对照之间无显著差异。此外, PDI基因沉默后, 稻米的透明度下降、垩白度增加, 但对籽粒粗蛋白总量和直链淀粉含量的影响不甚明显。